A Method for In Vitro Culture of Rat Zymbal Gland: Use in Mechanistic Studies of Benzene Carcinogenesis in Combination with 32 P-Postlabeling

Abstract

Zymbal glands were excised bilaterally from the ear ducts of female Sprague-Dawley rats (three/group), minced into approximately four fragments per gland, and transferred into a microtiter plate containing 1.5 mL per well of Waymouth's tissue culture medium supplemented with fetal calf serum, hydrocortisone, insulin, and gentamicin. After addition of a test compound or solvent vehicle, plates were incubated for 6, 24, 48, or 96 hr at 37°C in a humidified atmosphere of 5% CO2 in air. Tissue in culture for 6 hr was histologically indistinguishable from the freshly excised tissue, while that in culture for 24, 48, and 96 hr showed a progressive deterioration often with necrosis and/or squamous metaplasia. More pronounced deterioration was noted in samples treated with 750 or 1500 pg/mL of benzene. Using a nuclease Pi-enhanced 32P-postlabeling assay, aromatic DNA adducts were detected in cultured Zymbal glands exposed for 48 hr to benzene and its derivatives, as well as to 7,12-dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (AAF). Benzene produced very low levels of adducts (0.5 adducts per 109 nucleotides), whereas its congeners produced relatively high levels of adducts (50-2000 lesions per 109 nucleotides), which decreased in the order benzoquinone > hydroquinone > phenol > benzenetriol > catechol. Each adduct profile overall was characteristic for the compound studied, suggesting the formation of compound-specific electrophiles. AAF and DMBA adducts were identical to those formed in vivo in animals. Our results show that the Zymbal glands are capable of metabolizing different carcinogens to DNA-reactive intermediates, a process that may be causally associated with tumor formation in vivo in this organ.

ATSDR evaluation of health effects of benzene and relevance to public health

“…Furthermore, DNA adducts with benzene metabolites have been found after benzene exposure (Hedli, et al, 1991;Lutz and Schlatter, 1977;Reddy, et al, 1989). Ong, et al (1995) evaluated various biomarkers of benzene exposure for their relationship with environmental benzene levels.…”

“…Hemoglobin and albumin adducts of the benzene metabolites, benzene oxide and 1,4benzoquinone, have been used as biomarkers of exposure to benzene (Bechtold and Henderson, 1993;Bechtold, et al, 1992a,b;Smith and Rothman, 2000;Yeowell-O'Connell, et al, 1998, 2001. Furthermore, DNA adducts with benzene metabolites have been found after benzene exposure (Hedli, et al, 1991;Lutz and Schlatter, 1977;Reddy, et al, 1989). Ong, et al (1995) evaluated various biomarkers of benzene exposure for their relationship with environmental benzene levels.…”

“…Hedli, et al (1991) observed that in rats, benzene metabolite-DNA adducts were observed in the bone marrow at doses that did not affect bone marrow cellularity, and suggested that monitoring of the bone marrow DNA adducts might be a sensitive bioassay of genotoxic effects of benzene exposure. Work by other researchers also suggests that monitoring DNA adducts or products of DNA damage might be useful (Bodell, et al, 1993;Chen, et al, 1994;Lagorio, et al, 1994a;Reddy, et al, 1989).…”

Benzene and Leukemia

“…The result of covalent binding was inhibition of production of mtRNA. Reddy et al (1989) cultured rat Zymbal glands in the presence of benzene, p-benzoquinone, hydroquinone, phenol, benzenetriol, or catechol . Whereas benzene yielded very low levels of adducts (0.5 adducts/10 9 nucleotides), the metabolites produced from 50 to 2000 adducts/10 9 nucleotides, with p-benzoquinone the most active.…”